Water-elutability of nucleic acids from metal-chelate affinity adsorbents: enhancement by control of surface charge density.

Full Abstract

Immobilized metal affinity chromatography (IMAC) is widely used for purification of proteins, especially "hexahistidine-tagged" recombinant proteins. We previously demonstrated the application of IMAC to selective capture of nucleic acids, including RNA, selectively-denatured genomic DNA, and PCR primers through interactions with purine bases exposed in single-stranded regions. We also found that the binding affinity of nucleic acids for IMAC adsorbents can be increased several-fold by addition of 20 volume% of neutral additives such as ethanol or DMSO. In the present work, it is demonstrated that bound nucleic acids can be effectively eluted with water instead of the usual imidazole-containing competitive eluants, when the surface density of negative charges is enhanced by operation at alkaline pH, or by deliberate metal-underloading of the anionic chelating ligands. With enhanced negative surface charge density, nucleic acid adsorption can be made strongly dependent on the presence of adsorption-promoting additives and/or repulsion-shielding salts, and removal of these induces elution. Complete water-elutability is demonstrated for baker's yeast RNA bound to 10% Cu(II)- underloaded IDA Chelating Sepharose in a binding buffer of 20 mM HEPES, 240 mM NaCl, pH 7. Water elutability will significantly enhance the utility of IMAC in nucleic acid separations.Copyright 2006 John Wiley & Sons, Ltd.

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Author information

Author/s: Fu, Joseph Y (JY); Potty, Ajish S R (AS); Fox, George E (GE); Willson, Richard C (RC);

Affiliation: Department of Chemical Engineering, University of Houston, Houston, TX 77204-4004, USA.

Journal and publication information

Publication Type: Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.

Journal: Journal of molecular recognition : JMR (J Mol Recognit), published in England. (Language: eng)

Reference: -2006 Jul-Aug; vol 19 (issue 4) : pp 348-53

Dates: Created 2006/08/28; Completed 2006/09/29; Revised 2006/11/15;

PMID: 16865664, status: MEDLINE (last retrieval date: 12/26/2008)

Sourced from the National Library of Medicine. Abstract text and other information may be subject to copyright.

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MeSH headings (categories)

This article was linked to the MESH Headings shown below.

Adsorption
Chelating Agents - chemistry
Chromatography, Affinity
Copper - chemistry
Hydrogen-Ion Concentration
Imidazoles - chemistry
Imino Acids - chemistry

Ligands
Oligonucleotides - chemistry; isolation & purification
Purines - chemistry
RNA, Fungal - chemistry
Sepharose - chemistry
Surface Properties
Water - chemistry

Associated Chemicals: Chelating Agents (0) ; Imidazoles (0) ; Imino Acids (0) ; Ligands (0) ; Oligonucleotides (0) ; Purines (0) ; RNA, Fungal (0) ; iminodiacetic acid (142-73-4) ; imidazole (288-32-4) ; Copper (7440-50-8) ; Water (7732-18-5) ; Sepharose (9012-36-6)

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